Myosin VI is perhaps the most unconventional of unconventional myosins. It uses a number of unique mechanisms that are not well understood to accomplish processive movements of similar step sizes to myosin V, but of opposite directionality, for the purposes of anchoring and transporting within cells. Myosin VI can exist as a monomer or a dimer, the cellular significance of which is poorly understood. This project will utilize in vitro expression and functional assays, structural determinations and in vivo assays to attempt to further our understanding of how myosin VI functions and what these molecules do in a cell. These goals will be realized by addressing the following specific aims: Aim 1. What are the adaptations in the myosin VI converter that create a large and variable (but inherently plus-end directed) step size? Hypothesis: While the unique insert at the end of the myosin VI converter, insert 2, is solely responsible for the reversal of directionality of myosin VI, the myosin VI converter has additional adaptations that allow a unique pre-powerstroke state and create a large and variable step size. Aim 2. What is the nature and purpose of the unusual myosin VI lever arm + extension? Hypothesis: The large step size of myosin VI is in part due to the unusual movements and conformations of its converter, which creates a larger swing (powerstroke) than for myosin V. With this larger swing, a shorter lever arm can provide the same step size as the myosin V lever arm. Aim 3. Further probe the mechanism of head gating during processivity Hypothesis: The unique insert, insert 1, of myosin VI is responsible for slow ATP binding, which becomes still slower with reverse strain, while ADP has access to the nucleotide binding pocket. This allows gating of the lead head of a dimer, as well as anchoring of a dimer. Aim 4. How does myosin VI dimerize and does it function as a dimer or monomer in cells? (collaboration with Karen Avraham) Hypothesis: Myosin VI dimerization is regulated by cargo interactions. We propose that the regulated dimerization obviates the need to regulate motor activity.